This stain must be freshly prepared. METHOD.--The cultivations employed should be smear agar cultures, twelve to eighteen hours old if incubated at 37°C, twenty-four to thirty hours if incubated at 22°C. 1. Remove a very small quantity of the growth by means of the platinum spatula. 2. Emulsify it with a few cubic centimetres of distilled water in a watch-glass, by gently moving the spatula to and fro in the water. Do not rub up the growth on the side of the watch-glass. Some workers prefer to use tap water, others employ normal saline solution, but distilled water gives the best emulsion. 3. Spread a thin film of the emulsion on a newly flamed cover-slip, using no force, but rather _leading_ the drop over the cover-slip with the platinum loop. 4. Allow the film to dry in the air, properly protected from falling dust. 5. Fix by passing thrice through the Bunsen flame, holding the cover-slip whilst doing so by one corner between the finger and thumb. 6. Pour on the film as much of the mordant as the cover-glass will hold. Grasp the cover-slip with the forceps and hold it, high above the flame, until steam rises. Allow the steaming mordant to remain in contact with the film two minutes. 7. Wash well in water and dry carefully. 8. Pour on the film as much of the stain as the cover-glass will hold. Steam over the flame as before for two minutes. 9. Wash well in water. 10. Dry and mount. ~2. "Pitfield" Original Method.~-- (a) _Mordant._-- Tannic acid 1 gramme Water 10 c.c. (b) _Stain._-- Saturated aqueous solution of alum 10 c.c. Saturated alcoholic solution of gentian violet 1 c.c. Distilled water 5 c.c. Mix equal parts of a and b before using. 1. Prepare and fix the film in the manner described above. 2. Boil the mixture and immerse the cover-slip in it, whilst still hot, for one minute. 3. Wash in water. 4. Examine in water; if satisfactory, dry and mount in Canada balsam. ~3. MacCrorrie's Method.~-- _Mordant-Stain._-- Measure out and mix. Night blue, saturated alcoholic solution 10 c.c. Potash alum, saturated aqueous solution 10 c.c. Tannin, 10 per cent. aqueous solution 10 c.c. NOTE.--The addition of gallic acid, 0.1 to 0.2 gramme, may improve the solution, but is not necessary. METHOD.-- 1. Prepare and fix the films as above. 2. Pour some of the mordant-stain on the film and warm gently, high above the flame, for two minutes (or place in the "hot" incubator for a like period). 3. Wash thoroughly in water. 4. Dry and mount. ~4. Loeffler's Method.~-- (a) _Mordant._--